Screening ubiquitin specific protease activities using chemically synthesized ubiquitin and ubiquitinated peptides
2017
Abstract
Ubiquitin, a 76 amino acid protein, is a key component that contributes to cellular protein homeostasis. The specificity of this modification is due to a series of enzymes: ligases, attaching the
ubiquitinto a lysine, and deubiquitinases, which remove it. More than a hundred of such proteins are implicated in the regulation of
protein turnover. Their specificities are only partially understood. We chemically synthesized
ubiquitin, attached it to lysines belonging to the protein sequences known to be
ubiquitinated. We chose the model protein “murine
double minute2” (
mdm2), a
ubiquitin ligase, itself
ubiquitinatedand deubiquitinated. We folded the
ubiquitinatedpeptides and checked their tridimensional conformation. We assessed the use of these substrates with a series of fifteen deubiquitinases to show the potentiality of such an enzymological technique. By manipulating the sequence of the peptide on which
ubiquitinis attached, we were able to detect differences in the enzyme/substrate recognition, and to determine that these differences are deubiquitinase-dependent. This approach could be used to understand the substrate/protein relationship between the protagonists of this reaction. The methodology could be customized for a given substrate and used to advance our understanding of the key amino acids responsible for the deubiquitinase specificities.
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